Identifying PFAS hotspots in surface waters of South Carolina using a new optimized total organic fluorine method and target LC-MS/MS.

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants of concern due to their long persistence in the environment, toxicity, and widespread presence in humans and wildlife. Knowledge regarding the extent of PFAS contamination in the environment is limited due to the need for analytical methods that can reliably quantify all PFAS, since traditional target methods using liquid chromatography (LC)-mass spectrometry (MS) fail to capture many. For a more comprehensive analysis, a total organic fluorine (TOF) method can be used as a screening tool. We combined TOF analysis with target LC-MS/MS analysis to create a statewide PFAS hotspot map for surface waters throughout South Carolina. Thirty-eight of 40 locations sampled contained detectable concentrations of organic fluorine (above 100 ng/L). Of the 33 target PFAS analyzed using LC-MS/MS, the most prevalent were perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHpA), and perfluorohexanesulfonate (PFHxS). On average, LC-MS/MS only accounted for 2 % of the TOF measured. Locations with high TOF did not necessarily correlate to high total quantified PFAS concentrations and vice-versa, demonstrating the limitations of target PFAS analysis and indicating that LC-MS may miss highly contaminated sites. Results suggest that future surveys should utilize TOF to more comprehensively capture PFAS in water bodies.

What are the bottlenecks to health data sharing in Switzerland? An interview study.

BACKGROUND: While health data sharing for research purposes is strongly supported in principle, it can be challenging to implement in practice. Little is known about the actual bottlenecks to health data sharing in Switzerland. AIMS OF THE STUDY: This study aimed to assess the obstacles to Swiss health data sharing, including legal, ethical and logistical bottlenecks. METHODS: We identified 37 key stakeholders in data sharing via the Swiss Personalised Health Network ecosystem, defined as being an expert on sharing sensitive health data for research purposes at a Swiss university hospital (or a Swiss disease cohort) or being a stakeholder in data sharing at a public or private institution that uses such data. We conducted semi-structured interviews, which were transcribed, translated when necessary, and de-identified. The entire research team discussed the transcripts and notes taken during each interview before an inductive coding process occurred. RESULTS: Eleven semi-structured interviews were conducted (primarily in English) with 17 individuals representing lawyers, data protection officers, ethics committee members, scientists, project managers, bioinformaticians, clinical trials unit members, and biobank stakeholders. Most respondents felt that it was not the actual data transfer that was the bottleneck but rather the processes and systems around it, which were considered time-intensive and confusing. The templates developed by the Swiss Personalised Health Network and the Swiss General Consent process were generally felt to have streamlined processes significantly. However, these logistics and data quality issues remain practical bottlenecks in Swiss health data sharing. Areas of legal uncertainty include privacy laws when sharing data internationally, questions of "who owns the data", inconsistencies created because the Swiss general consent is perceived as being implemented differently across different institutions, and definitions and operationalisation of anonymisation and pseudo-anonymisation. Many participants desired to create a "culture of data sharing" and to recognise that data sharing is a process with many steps, not an event, that requires sustainability efforts and personnel. Some participants also stressed a desire to move away from data sharing and the current privacy focus towards processes that facilitate data access. CONCLUSIONS: Facilitating a data access culture in Switzerland may require legal clarifications, further education about the process and resources to support data sharing, and further investment in sustainable infrastructureby funders and institutions.

SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration.

High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. Here we show that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1ß release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in male mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1ß release, contributing to an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of male and female postmortem human brains demonstrate that SA is hyperactivated in Alzheimer's disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer's disease, providing mechanistic insight into the biology of neuroinflammation.

Predicting Medical Student Performance With a Situational Judgment Test in Admissions.

PURPOSE: To explore correlations between AAMC situational judgment test (SJT) scores, other admissions data, and learners' medical school performance. METHOD: First- and second-year medical students from 8 U.S. MD-granting medical schools completed a prototype version of the AAMC SJT in 2017. Outcomes included research-only faculty ratings of student performance, final course grades, and faculty evaluations of student performance, 2017-2018 and 2018-2019 academic years. Bivariate correlations were used to investigate the relationship between SJT scores and student performance outcomes and hierarchical regressions to investigate whether SJT scores provided incremental validity over MCAT total scores and cumulative undergraduate grade point averages (UGPAs) for predicting student performance outcomes. RESULTS: In general, there were small positive correlations with research-only faculty ratings from the first year of medical school, with the highest for social skills/service orientation ( rcorrected = .33, P < .05). Correlations were higher, with the highest for social skills/service orientation and cultural competence ( rcorrected = .33 and .36, respectively, P < .05) in the second year in medical school. SJT scores improved prediction of research-only faculty ratings over MCAT total scores and UGPAs for reliability and dependability/capacity for improvement, cultural competence, social skills/service orientation, and the overall composite score in the first year and for resilience and adaptability, social skills/service orientation, cultural competence, and the overall composite score in the second year. SJT scores demonstrated small correlations with course grades ( rsample-weighted = .10, P = ns) and faculty evaluations related to professionalism skills ( rsample-weighted = .14, P < .05); however, MCAT total scores explained most of the variance associated with course outcomes. CONCLUSIONS: These studies provide initial evidence that SJT scores may add value to the medical school admissions process because scores were related to faculty ratings of professional behaviors and provided unique information relative to MCAT scores and UGPAs.

Discovery of a Potent Proteolysis Targeting Chimera Enables Targeting the Scaffolding Functions of FK506-Binding Protein 51 (FKBP51).

The FK506-binding protein 51 (FKBP51) is a promising target in a variety of disorders including depression, chronic pain, and obesity. Previous FKBP51-targeting strategies were restricted to occupation of the FK506-binding site, which does not affect core functions of FKBP51. Here, we report the discovery of the first FKBP51 proteolysis targeting chimera (PROTAC) that enables degradation of FKBP51 abolishing its scaffolding function. Initial synthesis of 220 FKBP-focused PROTACs yielded a plethora of active PROTACs for FKBP12, six for FKBP51, and none for FKBP52. Structural analysis of a binary FKBP12:PROTAC complex revealed the molecular basis for negative cooperativity. Linker-based optimization of first generation FKBP51 PROTACs led to the PROTAC SelDeg51 with improved cellular activity, selectivity, and high cooperativity. The structure of the ternary FKBP51:SelDeg51:VCB complex revealed how SelDeg51 establishes cooperativity by dimerizing FKBP51 and the von Hippel-Lindau protein (VHL) in a glue-like fashion. SelDeg51 efficiently depletes FKBP51 and reactivates glucocorticoid receptor (GR)-signalling, highlighting the enhanced efficacy of full protein degradation compared to classical FKBP51 binding.

Large-scale, in-cell photocrosslinking at single-residue resolution reveals the molecular basis for glucocorticoid receptor regulation by immunophilins.

The Hsp90 co-chaperones FKBP51 and FKBP52 play key roles in steroid-hormone-receptor regulation, stress-related disorders, and sexual embryonic development. As a prominent target, glucocorticoid receptor (GR) signaling is repressed by FKBP51 and potentiated by FKBP52, but the underlying molecular mechanisms remain poorly understood. Here we present the architecture and functional annotation of FKBP51-, FKBP52-, and p23-containing Hsp90-apo-GR pre-activation complexes, trapped by systematic incorporation of photoreactive amino acids inside human cells. The identified crosslinking sites clustered in characteristic patterns, depended on Hsp90, and were disrupted by GR activation. GR binding to the FKBPFK1, but not the FKBPFK2, domain was modulated by FKBP ligands, explaining the lack of GR derepression by certain classes of FKBP ligands. Our findings show how FKBPs differentially interact with apo-GR, help to explain the differentiated pharmacology of FKBP51 ligands, and provide a structural basis for the development of improved FKBP ligands.

[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

Structure-Based Discovery of a New Selectivity-Enabling Motif for the FK506-Binding Protein 51.

In recent years, the selective inhibition of FKBP51 has emerged as a possible treatment for chronic pain, obesity-induced diabetes, or depression. All currently known advanced FKBP51-selective inhibitors, including the widely used SAFit2, contain a cyclohexyl residue as a key motif for enabling selectivity over the closest homologue and anti-target FKBP52. During a structure-based SAR exploration, we surprisingly discovered thiophenes as highly efficient cyclohexyl replacement moieties that retain the strong selectivity of SAFit-type inhibitors for FKBP51 over FKBP52. Cocrystal structures revealed that the thiophene-containing moieties enable selectivity by stabilizing a flipped-out conformation of Phe67 of FKBP51. Our best compound, 19b, potently binds to FKBP51 biochemically as well as in mammalian cells, desensitize TRPV1 in primary sensory neurons, and has an acceptable PK profile in mice, suggesting its use as a novel tool compound for studying FKBP51 in animal models of neuropathic pain.

Chemical Doping by Fluorination and Its Impact on All Energy Levels of π-Conjugated Systems.

Halogenation of organic molecules causes chemical shifts of C1s core-level binding energies that are commonly used as fingerprints to identify chemical species. Here, we use synchrotron-based X-ray photoelectron spectroscopy and density functional theory calculations to unravel such chemical shifts by examining different partially fluorinated pentacene derivatives. Core-level shifts occur even for carbon atoms distant from the fluorination positions, yielding a continuous shift of about 1.8 eV with increasing degree of fluorination for pentacenes. Since also their LUMO energies shift markedly with the degree of fluorination of the acenes, core-level shifts result in a nearly constant excitation energy of the leading π* resonance as obtained in complementary recorded K-edge X-ray absorption spectra, hence demonstrating that local fluorination affects the entire π-system, including valence and core levels. Our results thus challenge the common picture of characteristic chemical core-level energies as fingerprint signatures of fluorinated π-conjugated molecules.

Structural and biochemical insights into FKBP51 as a Hsp90 co-chaperone.

The FK506-binding protein 51 (FKBP51) is a high-molecular-weight immunophilin that emerged as an important drug target for stress-related disorders, chronic pain, and obesity. It has been implicated in a plethora of molecular pathways but remains best characterized as a co-chaperone of Hsp90 in the steroid hormone receptor (SHR) maturation cycle. However, the mechanistic and structural basis for the regulation of SHRs by FKBP51 and the usually antagonistic function compared with its closest homolog FKBP52 remains enigmatic. Here we review recent structural and biochemical studies of FKBPs as regulators in the Hsp90 machinery. These advances provide important insights into the roles of FKBP51 and FKBP52 in SHR regulation.

Hierarchical Structure of Cellulose Nanofibril-Based Foams Explored by Multimodal X-ray Scattering.

Structural characterization techniques are fundamental to correlate the material macro-, nano-, and molecular-scale structures to their macroscopic properties and to engineer hierarchical materials. Here, we combine X-ray transmission with scanning small- and wide-angle X-ray scattering (sSWAXS) to investigate ultraporous and lightweight biopolymer-based foams using cellulose nanofibrils (CNFs) as building blocks. The power of multimodal sSWAXS for multiscale structural characterization of self-assembled CNFs is demonstrated by spatially resolved maps at the macroscale (foam density and porosity), at the nanoscale (foam structural compactness, CNF orientation in the foam walls, and CNF packing state), and at the molecular scale (cellulose crystallite dimensions). Specifically, we compare the impact of freeze-thawing-drying (FTD) fabrication steps, such as static/stirred freezing and thawing in ethanol/water, on foam structural hierarchy spanning from the molecular to the millimeter scale. As such, we demonstrate the potential of X-ray scattering imaging for hierarchical characterization of biopolymers.

Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides.

Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein-ligand-bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets.

Permanent Dipole Moments Enhance Electronic Coupling and Singlet Fission in Pentacene.

Singlet fission (SF), the photophysical process in which one singlet exciton is transformed into two triplets, depends inter alia on the coupling of electronic states. Here, we use fluorination and the resulting changes in partial charge distribution across the chromophore backbone as a particularly powerful tool to control this parameter in pentacene. We find that the introduction of a permanent dipole moment leads to an enhanced coupling of Frenkel exciton and charge transfer states and to an increased SF rate which we probed using ultrafast transient absorption spectroscopy. These findings are contrasted with H-aggregate formation and a significantly reduced triplet-pair state lifetime in a fluorinated pentacene for which the different partial charge distribution leads to a negligible dipole moment.

Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion.

The stress response is an essential mechanism for maintaining homeostasis, and its disruption is implicated in several psychiatric disorders. On the cellular level, stress activates, among other mechanisms, autophagy that regulates homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway in which autophagosomes fuse with the plasma membrane rather than with lysosomes. Here, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy via the stress-responsive co-chaperone FK506-binding protein 51. We identify the matrix metalloproteinase 9 (MMP9) as one of the proteins secreted in response to stress. Using cellular assays and in vivo microdialysis, we further find that stress-enhanced MMP9 secretion increases the cleavage of pro-brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF). BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a cellular stress adaptation mechanism that bears the potential of opening avenues for the understanding of the pathophysiology of stress-related disorders.

Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with Enhanced Selectivity.

Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding protein 51 (FKBP51), which has emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs. FKBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During a macrocyclization pilot study, we observed that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with a new binding mode featuring a transient conformation, which is disfavored for the small FKBPs. Using a conformation-sensitive assay we show that this binding mode occurs in solution and is characteristic for this new class of compounds. The discovered macrocycles are non-immunosuppressive, engage FKBP51 in cells, and block the cellular effect of FKBP51 on IKKα. Our findings provide a new chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Development of NanoBRET-Binding Assays for FKBP-Ligand Profiling in Living Cells.

FK506-binding proteins (FKBPs) are promising targets for a variety of disorders and infectious diseases. High FKBP occupancy is thought to be necessary for ligands to effectively compete with the endogenous intracellular functions of FKBPs. Here, we report the development of NanoBRET assays for the most prominent cytosolic FKBPs, FKBP12, 12.6, 51 and 52. These assays allowed rapid profiling of FKBP ligands for target engagement and selectivity in living cells. These assays confirmed the selectivity of SAFit-type ligands for FKBP51 over FKBP52 but revealed a substantial offset for the intracellular activity of these ligands compared to bicyclic ligands or natural products. Our results stress the importance to control for intracellular FKBP occupancy and provide the assays to guide further FKBP ligand optimization.

Impact of fluorination on interface energetics and growth of pentacene on Ag(111).

We studied the structural and electronic properties of 2,3,9,10-tetrafluoropentacene (F4PEN) on Ag(111) via X-ray standing waves (XSW), low-energy electron diffraction (LEED) as well as ultraviolet and X-ray photoelectron spectroscopy (UPS and XPS). XSW revealed that the adsorption distances of F4PEN in (sub)monolayers on Ag(111) were 3.00 Å for carbon atoms and 3.05 Å for fluorine atoms. The F4PEN monolayer was essentially lying on Ag(111), and multilayers adopted π-stacking. Our study shed light not only on the F4PEN-Ag(111) interface but also on the fundamental adsorption behavior of fluorinated pentacene derivatives on metals in the context of interface energetics and growth mode.

Dual physically and chemically crosslinked regenerated cellulose - Gelatin composite hydrogels towards art restoration.

Art restoration poses many challenges for scientists and conservators, as any restorative action can lead to lasting modification or damage to the original artefact. Recent interest in gel encapsulation has grown due to the ability to control the cleaning action; yet the restoration of modern paints such as acrylic-based systems still presents issues due to their extremely high sensitivity to most solvents. Herein, the preparation of dual physically and chemically crosslinked hydrogels based on regenerated cellulose and cinnamoyl-modified gelatin is demonstrated. These dual crosslinked hydrogels show increased mechanical strength and enhanced water retention compared to pure physically crosslinked hydrogels. When applied to acrylic-based paint surfaces, the dual crosslinked hydrogels extract a smaller amount of hydrophilic additives (albeit still leading to swelling within the paint film) versus physically crosslinked gels. It is anticipated that this dual crosslinking approach can be broadly applied to prepare gels for conservation of cultural heritage artefacts.

Modulating the Electronic and Solid-State Structure of Organic Semiconductors by Site-Specific Substitution: The Case of Tetrafluoropentacenes.

The properties as well as solid-state structures, singlet fission, and organic field-effect transistor (OFET) performance of three tetrafluoropentacenes (1,4,8,11: 10, 1,4,9,10: 11, 2,3,9,10: 12) are compared herein. The novel compounds 10 and 11 were synthesized in high purity from the corresponding 6,13-etheno-bridged precursors by reaction with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate at elevated temperatures. Although most of the molecular properties of the compounds are similar, their chemical reactivity and crystal structures differ considerably. Isomer 10 undergoes the orbital symmetry forbidden thermal [4+4] dimerization, whereas 11 and 12 are much less reactive. The isomers 11 and 12 crystallize in a herringbone motif, but 10 prefers π-π stacking. Although the energy of the first electric dipole-allowed optical transition varies only within 370 cm-1 (0.05 eV) for the neutral compounds, this amounts to roughly 1600 cm-1 (0.20 eV) for radical cations and 1300 cm-1 (0.16 eV) for dications. Transient spectroscopy of films of 11 and 12 reveals singlet-fission time constants (91±11, 73±3 fs, respectively) that are shorter than for pentacene (112±9 fs). OFET devices constructed from 11 and 12 show close to ideal thin-film transistor (TFT) characteristics with electron mobilities of 2×10-3 and 6×10-2  cm2 V-1 s-1 , respectively.

Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays' and cross-validation with resections, in patients with, stage I-III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.